Behavior of human osteoblast-like cells in contact with electrodeposited calcium phosphate coatings

Calcium-deficient hydroxyapatite (Ca-def-HAP) thin films were elaborated on Ti6Al4V substrates by electrodeposition. The coatings exhibit two different morphologies and crystallinities. Human osteoblast-like cells (MG-63) were cultured on the surfaces of these materials; the cell content and viability were evaluated up to 28 days. The scanning electron microscopy and biological investigations showed cells with a normal morphology, good proliferation, and viability from 7 to 21 days. But after 28 days, the number of live cells decreases in both cases; however, this decrease is less important in the case of calcium phosphate (CaP) coating surface when compared with the control (cell culture plastic). The cells cultured on Ca-def-HAP coating exhibit more cellular extensions and extracellular matrix. RT-PCR for type I collagen, alkaline phosphatase, and osteocalcin studies were also carried out, and was found that the CaP enhances gene expression of ALP and OC and thus the differentiation of osteoblast-like cells. Moreover, this study shows that the difference in the morphology of CaP coatings has no effect on the biocompatibility.     

Development of biodegradable polyurethane and bioactive glass nanoparticles scaffolds for bone tissue engineering applications

The development of polymer/bioactive glass has been recognized as a strategy to improve the mechanical behavior of bioactive glass-based materials. Several studies have reported systems based on bioactive glass/biopolymer composites. In this study, we developed a composite system based on bioactive glass nanoparticles (BGNP), obtained by a modified St?ber method. We also developed a new chemical route to obtain aqueous dispersive biodegradable polyurethane. The production of polyurethane/BGNP scaffolds intending to combine biocompatibility, mechanical, and physical properties in a material designed for tissue engineering applications. The composites obtained were characterized by structural, biological, and mechanical tests. The films presented 350% of deformation and the foams presented pore structure and mechanical properties adequate to support cell growth and proliferation. The materials presented good cell viability and hydroxyapatite layer formation upon immersion in simulated body fluid.     

Osteoblast interactions with calcium phosphate ceramics modified by coating with type I collagen

Complications associated with the use of autogenous bone in the repair or replacement of tissue lost through injury or disease have driven the search for alternative sources of graft material. Bioceramics containing hydroxyapatite (HA), tricalcium phosphate (TCP), or composites that combine the best properties of both of these materials are among the principal candidates. In this study, we have investigated the in vitro proliferation, morphology, and viability of an immortalized rat osteoblast cell line cultured on HA, TCP, and composites of the two in the ratios 75:25 (H75), 50:50 (H50), and 25:75 (H25) for 28 days. The biocompatibility of each material was examined in the presence and absence of a collagen coating. With the exception of H50, cell proliferation, quantified by carboxyfluorescein fluorescence, was enhanced by collagen coating of all materials for the first 14 days, although at later time points cell numbers were unaffected. It is notable that the collagen coating was least stable on H50, the only material not to show enhancement of cell growth on coating. Confocal laser scanning microscopy confirmed that cell growth was more extensive on coated materials over the first 7-14 days in culture, and the development of cell extensions and bridges across the pores in the materials was observed. Results indicate that collagen coating of calcium phosphate ceramics may also increase their compatibility and osseointegration in vivo.     

Cytocompatibility and response of osteoblastic-like cells to starch-based polymers: effect of several additives and processing conditions

This work reports on the biocompatibility evaluation of new biodegradable starch-based polymers that are under consideration for use in orthopaedic temporary applications and as tissue engineering scaffolds. It has been shown in previous works that by using these polymers it is both possible to produce polymer/hydroxyapatite (HA) composites (with or without the use of coupling agents) with mechanical properties matching those of the human bone, and to obtain 3D structures generated by solid blowing agents, that are suitable for tissue engineering applications. This study was focused on establishing the influence of several additives (ceramic fillers, blowing agents and coupling agents) and processing methods/conditions on the biocompatibility of the materials described above. The cytotoxicity of the materials was evaluated using cell culture methods, according to ISO/EN 109935 guidelines. A cell suspension of human osteosarcoma cells (HOS) was also seeded on a blend of corn starch with ethylene vinyl alcohol (SEVA-C) and on SEVA-C/HA composites, in order to have a preliminary indication on cell adhesion and proliferation on the materials surface. In general, the obtained results show that all the different materials based on SEVA-C, (which are being investigated for use in several biomedical applications), as well as all the additives (including the novel coupling agents) and different processing methods required to obtain the different properties/products, can be used without inducing a cytotoxic behaviour to the developed biomaterials.     

Biological evaluation of nano-hydroxyapatite-zirconia (HA-ZrO2) composites and strontium-hydroxyapatite (Sr-HA) for load-bearing applications

The biological response of strontium (Sr) doped hydroxyapatite (HA) and hydroxyapatite-zirconia (HA-ZrO?) composites produced by employing sol-gel technology, minimal ZrO? loadings, and novel microwave-sintering regimes thereby retarding decomposition, is reported. In vitro evaluations indicate that all materials induce a favorable response from rat osteosarcoma cells. In vivo evaluations show osteoconductivity and biocompatibility for both the Sr-HA and HA-ZrO?. The materials did not cause any inflammatory response in bone. The Sr-HA displays better biocompatibility which may be due to the incorporation of Sr and the formation of a surface apatite layer.     

Tensile properties,tension-tension fatigue and biological response of polyetheretherketone-hydroxyapatite composites for load-bearing orthopedic implants

Polyetheretherketone-hydroxyapatite composites were developed as alternative materials for load-bearing orthopedic applications. The amount of hydroxyapatite (HA) incorporated into the polyetheretherketone (PEEK) polymer matrix ranges from 5 to 40 vol% and these materials were successfully fabricated by injection molding. This study presents the mechanical and biological behavior of the composite materials developed. It was found that the amount of HA in the composite influenced the tensile properties. Dynamic behavior under tension-tension fatigue revealed that the fatigue-life of PEEK-HA composites were dependent on the HA content as well as the applied load. The biological responses of PEEK-HA composites carried out in vivo verified the biocompatibility and bioactive nature of the composite materials.     

碳化硅对成骨细胞增殖和分化的影响

目的研究碳化硅(SiC)的体外生物相容性,为探索SiC作为骨缺损修复材料的可行性奠定基础。方法实验组为实质SiC,对照组为致密羟基磷灰石(HA)。将取自大鼠胎鼠颅骨的成骨细胞分别接种于2组材料表面。分别采用扫描电子显微镜、四甲基偶氮唑盐(MTT)法、碱性磷酸酶(ALP)含量测定技术、流式细胞仪检测细胞周期,观察分析两种材料表面上的细胞的增殖和分化能力。结果扫描电子显微镜显示:原代成骨细胞在两种材料表面伸展良好,细胞在材料表面伸出很多伪足,并且在材料表面可见明显的细胞外基质沉积。两组材料上细胞的MTT值从1、3、5、7 d随观察时间逐渐升高,并且各个时间点实质SiC均高于致密HA,两者之间的差异有统计学意义(P<0.05)。两组材料上细胞的ALP含量从1、3、5、7 d随观察时间逐渐升高;在1、3 d时,致密HA高于实质SiC,在5、7 d时,致密HA低于实质SiC,但是两组之间的差异均无统计学意义(P>0.05)。1、3、5 d两组材料的细胞增值指数(PI)值逐渐升高,高峰出现在第5天,第7天时下降,两组材料在各个时间点的PI均接近,差异无统计学意义(P>0.05)。结论实质SiC具有与致密HA相似的良好的细胞相容性。     

仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原的制备及其细胞相容性

背景：目前骨组织工程常用的支架材料主要有无机材料、有机高分子材料及天然衍生材料等,上述材料各有优缺点,为了充分发挥各类材料的优势,弥补其不足,目前多采用联合材料制备复合支架。 目的：制备新型仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原,并观察其对骨髓间充质干细胞增殖、黏附及分化的影响。 方法：制备壳聚糖/纳米羟基磷灰石/胶原复合支架材料,扫描电镜观察支架材料表面微观形貌;采用真空吸附法将骨形态发生蛋白7多肽与支架材料复合,高效液相色谱仪检测骨形态发生蛋白7多肽在体外的释放规律;将骨髓间充质干细胞接种到复合骨形态发生蛋白7多肽的仿生支架材料上,以未复合多肽的支架材料作为对照,检测支架材料表面细胞增殖、黏附率、生长形态及碱性磷酸酶活性。 结果与结论：壳聚糖/纳米羟基磷灰石/胶原支架材料呈多孔状,孔径10~100 µm;骨形态发生蛋白7多肽可以从支架材料中缓慢释出;在复合多肽的仿生支架材料表面,骨髓间充质干细胞的黏附及向成骨细胞方向分化能力均明显强于对照组(P < 0.05),而增殖能力与对照组差异无显著性意义(P > 0.05)。说明新型仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原是一种理想的骨组织工程支架材料,具有良好的细胞相容性。     

Evaluation of metal implants coated with several types of ceramics as biomaterials

The in vivo biocompatibility of metals coated with several different types of ceramics [alumina (alpha-Al2O3), titanium oxide (TiO2), titanium nitride (TiN), and hydroxyapatite (HAP)] was investigated. These composites had been devised for the purpose of incorporation into the stem of a total hip prosthesis. The materials were inserted into the mid-diaphyseal region of the femurs of adult dogs, and follow-up quantitative histological comparisons were performed for a period of up to 96 weeks. HAP-coated composites showed the best biocompatibility.     

In vitro evaluation of novel bioactive composites based on Bioglass-filled polylactide foams for bone tissue engineering scaffolds

Highly porous poly(DL-lactic acid) (PDLLA) foams and Bioglass-filled PDLLA composite foams were characterized and evaluated in vitro as bone tissue engineering scaffolds. The hypothesis was that the combination of PDLLA with Bioglass in a porous structure would result in a bioresorbable and bioactive composite, capable of supporting osteoblast adhesion, spreading and viability. Composite and unfilled foams were incubated in simulated body fluid (SBF) at 37 degrees C to study the in vitro degradation of the polymer and to detect hydroxyapatite (HA) formation, which is a measure of the materials' in vitro bioactivity. HA was detected on all the composite samples after incubation in SBF for just 3 days. After 28 days immersion the foams filled with 40 wt % Bioglass developed a continuous layer of HA. The formation of HA for the 5 wt % Bioglass-filled foams was localized to the Bioglass particles. Cell culture studies using a commercially available (ECACC) human osteosarcoma cell line (MG-63) were conducted to assess the biocompatibility of the foams and cell attachment to the porous substrates. The osteoblast cell infiltration study showed that the cells were able to migrate through the porous network and colonize the deeper regions within the foam, indicating that the composition of the foams and the pore structures are able to support osteoblast attachment, spreading, and viability. Rapid formation of HA on the composites and the attachment of MG-63 cells within the porous network of the composite foams confirms the high in vitro bioactivity and biocompatibility of these materials and their potential to be used as scaffolds in bone tissue engineering and repair.     

羟基磷灰石/壳聚糖生物复合材料的制备研究进展

羟基磷灰石/壳聚糖复合材料因其生物相容性和合适的力学性能逐渐成为骨替代材料研究的热点。本文综述了羟基磷灰石/壳聚糖复合材料的研究现状,探讨了其特点、制备和性能。并在此基础上提出了此类材料今后的发展方向：三相复合材料和电、磁学性能的研究。     

Laser microfabrication of hydroxyapatite-osteoblast-like cell composites

We have developed a novel approach for layer-by-layer growth of tissue-engineered materials using a direct writing process known as matrix assisted pulsed laser evaporation direct write (MAPLE DW). Unlike conventional cell-seeding methods, this technique provides the possibility for cell-material integration prior to artificial tissue fabrication. This process also provides greater flexibility in selection and processing of scaffold materials. In addition, MAPLE DW offers rapid computer-controlled deposition of mesoscopic voxels at high spatial resolutions. We have examined MAPLE DW processing of zirconia and hydroxyapatite scaffold materials that can provide a medical device with nearly inert and bioactive implant-tissue interfaces, respectively. We have also demonstrated codeposition of hydroxyapatite, MG 63 osteoblast-like cells, and extracellular matrix using MAPLE DW. We have shown that osteoblast-like cells remain viable and retain the capacity for proliferation when codeposited with bioceramic scaffold materials. Our results on MG 63-hydroxyapatite composites can be extended to develop other integrated cell-scaffold structures for medical and dental applications.     

Materials from Titanium—Cobalt Alloys for Hybrid Implants

We proposed a new method to increase the biocompatibility of porous materials that were synthesized from titanium and cobalt allows by the method of self-propagating high-temperature synthesis. This method suggested the introduction of calcium hydroxyapatite into the reaction mixture. Administration of calcium hydroxyapatite into the reaction mixture had a modifying effect on the structure and surface of the pore space and biocompatibility of composite materials. Administration of calcium hydroxyapatite crystals was followed by a significant decrease in the size of pores and appearance of water-soluble fractions, which inhibited the activity of cells. However, treatment with amorphous nanodispersed calcium hydroxyapatite increased the biocompatibility and adhesiveness of materials for mesenchymal stem cells. The pore space and mechanical characteristics of materials obtained with amorphous nanodispersed calcium hydroxyapatite were similar to the properties of natural bone. Moreover, these materials surpassed titanium—cobalt allows in biocompatibility. Our results indicate that the introduction of amorphous nanodispersed calcium hydroxyapatite into the reaction mixture during self-propagating high-temperature synthesis has a modifying effect on the pore space of composite materials and increases their biocompatibility and adhesiveness for cells. We conclude that these materials may be used as a carrier of stem cells and progenitor cells in hybrid implants. Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 52–58, 2009     

Biodegradable polymer/hydroxyapatite composites: surface analysis and initial attachment of human osteoblasts

Biodegradable polymer/hydroxyapatite (HA) composites have potential application as bone graft substitutes. Thin films of polymer/HA composites were produced, and the initial attachment of primary human osteoblasts (HOBs) was assessed to investigate the biocompatibility of the materials. Poly(epsilon-caprolactone) (PCL) and poly(L-lactic acid) (PLA) were used as matrix materials for two types of HA particles, 50-microm sintered and submicron nonsintered. Using ESEM, cell morphology on the surfaces of samples was investigated after 90 min, 4 h, and 24 h of cell culture. Cell activity and viability were assessed after 24 h of cell culture using Alamar blue and DNA assays. Surface morphology of the polymer/HA composites and HA exposure were investigated using ESEM and EDXA, respectively. ESEM enabled investigation of both cell and material surface morphology in the hydrated condition. Combined with EDXA it permitted chemical and visual examination of the composite. Differences in HA exposure were observed on the different composite surfaces that affected the morphology of attached cells. In the first 4 h of cell culture, the cells were spread to a higher degree on exposed HA regions of the composites and on PLA than they were on PCL. After 24 h the cells were spread equally on all the samples. The cell activity after 24 h was significantly higher on the polymer/HA composites than on the polymer films. There was no significant difference in the activity of the cells on the various composite materials. However, cells on PCL showed higher activity compared to those on PLA. A polymer surface exhibiting "point exposure" of HA appeared to provide a novel and favorable substrate for primary cell attachment. The cell morphology and activity results indicate a favorable cell/material interaction and suggest that PLA and PCL and their composites with HA may be candidate materials for the reconstruction of bony tissue. Further investigations regarding long-term biomaterial/cell interactions and the effects of acidic degradation products from the biodegradable polymers are required to confirm their utility.     

聚醚醚酮-羟基磷灰石-碳纤维复合材料的体外细胞相容性研究

将大鼠成骨细胞与聚醚醚酮-羟基磷灰石-碳纤维(PEEK-HA-CF)复合材料间接和直接培养,通过CCK-8对复合材料的细胞毒性进行评价;流式细胞仪测定材料对细胞增殖指数的影响;测定复合培养后成骨细胞的碱性磷酸酶活性变化;应用扫描电镜(SEM)观察成骨细胞形态学变化。结果表明PEEK-HA-CF复合材料无细胞毒性,复合材料组胞增殖指数与空白对照组比无差异,3d时高于钛合金组。第7d时,PEEK-HA-CF组的碱性磷酸酶活性升高。SEM下,细胞在复合材料上生长良好。PEEK-HA-CF复合材料具有良好的细胞相容性,有希望成为一种新的骨科植入材料。     

Effect of hydroxyapatite as a component of biostable composites on population and proliferation of mesenchymal stem cells

Three groups of biostable composite materials were studied. The initial binder polymers (polymethylmethacrylate, polyamide-12, superhigh-molecular-weight polyethylene) and hydroxyapatite-containing composites on the basis of these polymers were tested. Biostable polymers, including those containing hydroxyapatite, were nontoxic for fibroblasts and mesenchymal stem cells: the adhesion parameters for these cells were maximum for polyamide-12 and superhigh-molecular-weight polyethylene and did not depend on the presence of hydroxyapatite. Cell adhesion to “ pure” polymethylmethacrylate was significantly lower than to other composites, but increased after integration of hydroxyapatite. The efficiency of proliferation of fibroblast and mesenchymal stem cell on the surface of polyamide-12 and superhigh-molecular-weight polyethylene was maximum and did not depend on the presence of hydroxyapatite. The efficiency of cell proliferation on the surface of “pure” polymethylmethacrylate was low, but increased significantly if it was combined with hydroxyapatite, particularly in areas of mineral particles accumulation. It seems that the presence of high amounts of hydroxyapatite in polymethylmethacrylate samples promotes cell adhesion and proliferation. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 2, pp. 83–87, April, 2007     

Synthesis, characterization of calcium phosphates/polyurethane composites for weight-bearing implants

Calcium phosphate (CaP)/polymer composites have been studied as an alternative graft material for the treatment of bone defects. In this study, lysine-triisocyanate-based polyurethane (PUR) composites were synthesized from both hydroxyapatite (HA) and β-tricalcium phosphate (TCP) to reduce the brittleness of CaP and increase the bioactivity of the polymer. The mechanical properties and in vitro cellular response were investigated for both HA/PUR and TCP/PUR composites. The composites were implanted in femoral defects in rats, and in vivo bioactivity was evaluated by X-rays, micro-computed tomography (μCT), and histological sections. In biomechanical testing, PUR improved the mechanical properties of the CaP, thus rendering it potentially suitable for weight-bearing applications. In vitro cell culture studies showed that CaP/PUR composites are biocompatible, with β-TCP enhancing the cell viability and proliferation relative to HA. CaP/PUR composites also supported the differentiation of osteoblastic cells on the materials. When implanted in rat femoral defects, the CaP/PUR composites were biocompatible and osteoconductive with no adverse inflammatory response, as evidenced by X-rays, μCT images, and histological sections. Additionally, a histological examination showed evidence of cellular infiltration and appositional remodeling. These results suggest that CaP/PUR composites could be potentially useful biomaterials for weight-bearing orthopaedic implants.     

Keratin-hydroxyapatite composites: biocompatibility, osseointegration, and physical properties in an ovine model

Reconstituted keratin has shown promise as an orthopaedic biomaterial. This in vivo study investigates the biological response of composite materials prepared from reconstituted keratin containing a high content of hydroxyapatite (HA) (40 wt % HA), implanted for up to 18 weeks in the long bones of sheep. Keratin-HA composites were compared with a commercially available polylactic acid (PLA) HA composite (BIO RCI HA?, Smith and Nephew). Porous keratin-HA materials displayed excellent biocompatibility and osseointegration, with full integration into bone by 12 weeks. Dense keratin-HA materials also showed excellent biocompatibility, with a more limited osseointegration, involving the penetration of new bone into the periphery of the implant after eight weeks. In contrast, the PLA-HA implant did not integrate with surrounding tissue. Microindentation showed that porous keratin-HA implants were initially soft, but became stiffer as new bone penetrated the implant from four weeks onwards. In contrast, although the initial rigidity of dense keratin-HA composites was maintained for at least two weeks, the implant material weakened after four weeks. The PLA-HA implant maintained its physical properties throughout the course of the trial. This study demonstrates the increased osseointegration/osteoconduction capacity of keratin-HA composites and provides further evidence supporting the suitability of keratin-based materials, such as bone graft substitutes and soft tissue fixation devices.     

Synthesis and characterization of wool keratin/hydroxyapatite nanocomposite

Taking the inspiration from the biomineral, the wool keratin was selected to modulate the assembly of nanosized hydroxyapatite (HA) crystals via a coprecipitation method. A series of keratin/HA nanocomposite with different ratios were synthesized by adjusting the concentrations of keratin solutions and calcium phosphate and their final components were detected by thermogravimetric analysis (TGA). The transmission electron microscopy (TEM) and X-ray diffraction (XRD) confirmed that keratin in the composite decreased the crystallinity of HA. Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to examine the chemical and surface structure of the composites. In vitro biocompatibility results revealed that cells showed better viability on keratin/HA composites which have a ratio of organics and inorganics similar to that of natural bones.